Novel calcium-independent phospholipases a2, genes thereof and promoter of the same

ABSTRACT

Novel calcium-independent phospholipases A 2 ; genes encoding the same; an antibody against them; an inherent promoter or a regulator gene which comprises a base sequence occurring in intron and inducing site-specific expression in response to an external stimulus; a method of expressing a target protein in response to an external stimulus; and an organism having this gene transferred thereinto. Novel calcium-independent phospholipases A 2  having an amino acid sequence represented by SEQ ID NO: 1, 3 or 5 or an amino acid sequence derived from such an amino acid sequence by the substitution, deletion or addition of one or more amino acids; a gene having a base sequence occurring in an intron and being capable of initiating RNA transcription due to an external stimulus such as a stimulus with kainic acid or an electrical stimulus; a method of regulating expression by using the gene; and an organism having the gene transferred thereinto.

TECHNICAL FIELD

[0001] The present invention relates to a calcium-independent novelphospholipase A2 (having a phospholipase A1 activity as well) and, moreparticularly, it relates to a novel phospholipase A2 which is acalcium-independent phospholipase A2, is a phospholipase A2 expressedspecifically in hippocampus by an external stimulation such asstimulation by kainic acid or electric stimulation and has an amino acidsequence described in SEQ ID NO: 1, NO: 5 or NO: 8 of the SequenceListing or an amino acid sequence where one or more amino acid(s) in theamino acid sequence is/are substituted with other amino acid(s) ordeficient or one or more amino acid(s) is/are added thereto.

[0002] The present further relates to gene having a base sequenceexisting in intron where the base sequence is able to make theinitiation of transcription of RNA specifically to hippocampal dentategyrus by external stimulation such as kainic acid stimulation orelectric stimulation possible, to a method for regulating the expressionusing the same and to a living thing into which the same is introduced.

BACKGROUND OF THE INVENTION

[0003] In the gene of eukaryotes, there are many cases where geneticinformation stipulating the amino acid sequence of protein isinterrupted. A moiety having the genetic information of amino acidsequence of protein is called exon while a moiety having no geneticinformation of amino acid sequence is called intron. After an mRNAprecursor is formed by a transcription of genetic DNA, it is subjectedto a splicing so that an intron moiety is cut off whereby mature mRNA isresulted.

[0004] It has not been clarified yet why such an intron moiety ispresent in eukaryotes. However, it has been presumed that, in manycases, one exon is coded as a specific domain (functioning region) ofprotein and, when new protein having the same function is needed duringthe process of development, necessary protein is able to be produced bya combination of different exons.

[0005] With regard to a splicing of the mRNA precursor before beingsubjected to the splicing, there has been also known the case where notonly intron is cut off but also exon moiety is cut off to give mRNAcoding for different protein having the function of the same type.

[0006] For example, calcitonin gene has six exons—A, B, C, D, calcitoninCCP and CGRP (calcitonin gene related peptide). Exon A and exon B arenon-translated region while translated region is other four exons. Whena transcription is carried out in nucleus of cell, all exons areincluded but a process of the splicing varies depending upon organs. Forexample, in thyroid C cells, exon of the sixth CGRP is also spliced and,as a result, protein of the translated product becomes a peptidecomprising C-D-calcitonin CCP mainly exhibiting an action of reducing aserum Ca. In hypothalamic cells, exon of the fifth calcitonin CCP isalso spliced and, as a result, protein of the translated product becomesa peptide comprising C-D-CGRP mainly playing a role of regulation ofpain and autonomous activity.

[0007] When an exon moiety is divided into some as such, it is possibleto produce different proteins where several exons are bonded ifnecessary. Although it has been explained to divide an exon for such apurpose, there has been almost no clarification yet for the necessity ofintron except the preparation of an exon moiety. It has been known thatmany of introns have sequences of 5′-GT and AG-3′ at the terminals andthat there is an intermediate region abundant in pyrimidine and it hasbeen believed that a splicing is carried out by recognizing thosesequences at both terminals.

[0008] Phospholipase A2 is widely distributed in mammals and microbesand it is mostly a membrane-bound enzyme and participates in metabolismof membrane phospholipids. A cytosolic phospholipase A2 (cPLA2α) of 85kDa is a kind of phospholipase A2 and cuts out arachidonic acid mostlyfrom membrane phospholipids producing physiologically active substancesby arachidonic acid cascades such as prostaglandin, thromboxane,leukotriene, etc. derived from arachidonic acid. It has been also knownthat the liberated arachidonic acid participates in various nervousfunctions in the brain and, until now, the present inventors have shownby a northern blot technique and an in situ hybridization that cPLA2α isabundantly expressed in cranial nerve cells.

[0009] On the other hand, kainic acid is a kind of amino acid and hasbeen isolated as an anthelmintic component in Digenea simplex. Sincekainic acid has a chemical structure similar to glutamic acid, it hasbeen known as a substance binding to a glutamic acid receptor in thebrain and the nerve cells of animals resulting in a neuron excitingaction.

[0010] In order to check the function of phospholipase A2 in the brain,the present inventors have applied kainic acid stimulation or electricstimulation thereto and found a novel phospholipase A2 (455 amino acids;molecular weight: about 50 K) which transiently expresses being limitedto dentate gyrus of hippocampus. This enzyme is a partial proteininitiating from the 308th methionine of a cytosolic phospholipase A2α(85 K) and, since it also expresses in genetically defective mouse ofthe said enzyme, it contains a specific promoter which site-specificallyexpresses in response to stimulation. Although this enzyme is notpresent under a non-stimulated state, it is expressed by electricstimulation and kainic acid stimulation and, unlike the conventionalphospholipase A2, it is independent upon calcium unlike the conventionalphospholipase A2, produces eicosanoid, regulates a cerebral function andparticipates in denaturation, apoptosis and regeneration of nerve cellswhereby it is believed to be a molecule holding the key to thosecerebral functions.

[0011] Further, this novel phospholipase A2 (455 amino acids; molecularweight: about 50 K) is a partial protein initiating from the 308thmethionine of a known cytosolic phospholipase A2α (85 K), a promoterregion specific for expressing this protein is present in the intronmoiety immediately before that and the present inventors have foundthat, in the intron, there is an intron having a function of making theinitiation of transcription of RNA possible. Under a usual state, thisintron has no function of initiating the transcription of RNA. However,when a certain condition is set, it has a function of initiating thetranscription of RNA not from the inherent transcription position butfrom the moiety of the base sequence of this intron.

DISCLOSURE OF THE INVENTION

[0012] The present invention provides a calcium-independent novelphospholipase A2 (455 amino acids; molecular weight: about 50 K), genecoding therefor and antibody against that.

[0013] The present invention further provides an intrinsic promoter orregulatory gene comprising a base sequence existing in intron andsite-specifically expressing in response to external stimulation. Thepresent invention furthermore provides a method for expressing a desiredprotein in response to an external stimulation and to a living thinginto which that is introduced.

[0014] The present invention still further provides a method forspecifically investigating the nerve stem cells since the KIDS cPLA2 ofthe present invention is specifically expressed in nerve stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a picture (as a substitute for a drawing) showing theresult of a northern blotting using various sites of cPro Leu Ala 2 asprobes. The upper column of FIG. 1 shows a base sequence of cPro Leu Ala2. Ala, B, Cys and Asp show the probes. The middle column of FIG. 1 isthe case where no kainic acid treatment is carried out (Lys Ala (−)) andthe lower column of FIG. 1 is the case of after 3 hours from the kainicacid treatment (Lys Ala(+), 3 h).

[0016]FIG. 2 is a picture (as a substitute for a drawing) showing theresult where hippocampus and cerebellum were subjected to a northernblotting with a lapse of time. The left side of FIG. 2 is hippocampuswhile the right side thereof is cerebellum. In each of them are shownthe blots after 0 hour, 0.5 hour, 3 hours, 8 hours, 14 hours and 18hours from the kainic acid treatment.

[0017]FIG. 3 is a picture (as a substitute for a drawing) showing theresult of an in situ hybridization in the brain of rat. The left side ofFIG. 3 is the result of a cross section of the brain while the rightside thereof is that from a vertical section of the brain. In FIG. 3,the parts which are in black are colored parts.

[0018]FIG. 4 is a picture (as a substitute for a drawing) where the partof dentate gyrus of hippocampus in the result of FIG. 3 is enlarged. Theleft side of FIG. 4 is the result where no kainic acid treatment wascarried out while the right side thereof is that after 3 hours from thekainic acid treatment.

[0019]FIG. 5 is a picture (as a substitute for a drawing) showing theresult of confirmation of expression of the desired protein by animmunohistochemical analysis using an antibody specifically recognizingLys Ile Asp Ser cPro Leu Ala 2 of the present invention. The left sideof the upper column of FIG. 5 is the case where no kainic acid treatmentwas carried out while the right side thereof is the case of 3 hoursafter the kainic acid treatment. The left side of the lower column ofFIG. 5 is the control where no treatment with an anti-Lys Ile Asp SercPro Leu Ala 2 antibody (Ile gGly) was carried out. The right side ofthe lower column of FIG. 5 is the result of chromatography in theabsence of an anti-Lys Ile Asp Ser cPro Leu Ala 2 antibody (Ile gGly)after 3 hours from the kainic acid treatment ((−) at the left side ofthe right side, lower column, FIG. 5) and in the presence of that ((+)at the right side thereof).

[0020]FIG. 6 is a picture (as a substitute for a drawing) showing theresult of investigation of expression after integration of cAsp Asn Alacoding for cPro Leu Ala 2α and Lys Ile Asp Ser cPro Leu Ala 2 of thepresent invention with an expression vector pThr racer Glu Phe. In FIG.6, the lane 1 is the case of a control vector, the lane 2 is the case ofcPro Leu Ala 2α/pThr racer Glu Phe and the lane 3 is the case of Lys IleAsp Ser cPro Leu Ala 2/pThr racer Glu Phe. In FIG. 6, the left side isthe case where an anti-Val 5 epitope Ile gGly was used, the middle isthe case where an anti-cPro Leu Ala 2α Ile gTyr was used and the rightside is the case where an anti-Lys Ile Asp Ser cPro Leu Ala 2Ile gGlywas used.

[0021]FIG. 7 shows the result of enzymatic activity of Lys Ile Asp SercPro Leu Ala 2 and cPro Leu Ala 2α of the present invention. In FIG. 7,the left side is that for Lys Ile Asp Ser cPro Leu Ala 2 of the presentinvention while the right side is that for cPro Leu Ala 2α. There wereused 1-Pro am-2-[¹⁴Cys] arachidonoyl-Pro Cys (black dots ()), 1-Proam-2-[¹⁴Cys]linoleoyl-Pro Cys (black triangles (▾)), 1-Proam-2-[¹⁴Cys]oleoyl-Pro Cys (black squares (▪)) and 1-Proam-2-[¹⁴Cys]palmitoyl-Pro Cys (asterisks (*)) as the substrates.

[0022]FIG. 8 shows the result of the test of calcium dependency of LysIle Asp Ser cPro Leu Ala 2 and cPro Leu Ala 2α of the present inventionon enzymatic activity using 1-Pro am-2-[¹⁴Cys]arachidonoyl-Pro Cys as asubstrate. The solid lines in FIG. 8 are the case of Lys Ile Asp SercPro Leu Ala 2 of the present invention while the broken lines thereinare the case of cPro Leu Ala 2α. In each of them, the black dots () arethe data in the absence of Glu Asp Thr Ala-Cys a while the open circles(◯) are those in the presence of Glu Asp Thr Ala-Cys a.

[0023]FIG. 9 shows the result given in the above FIG. 8 in terms of arelative ratio.

[0024]FIG. 10 is a picture (as a substitute for a drawing) showing theresult of investigation of expression of Lys Ile Asp Ser cPro Leu Ala 2of the present invention in a mouse defective of cPro Leu Ala. In FIG.10, the upper column is that for (+/+) of a knockout mouse while thelower column is that for (−/−) of a knockout mouse. In FIG. 10, the leftside is that where no kainic acid treatment was carried out (Lys Ala(−)) while the right side is that after 3 hours from the kainic acidtreatment (Lys Ala (+)).

[0025]FIG. 11 illustrates the state of expression of cPro Leu Ala 2 andLys Ile Asp Ser cPro Leu Ala 2. In FIG. 11, the upper columnschematically shows exon and intron of cPro Leu Ala 2 in genomic gene.

[0026]FIG. 12 shows a numbering for a base sequences from the first baseof intron immediately before exon containing “Met-308” of rat (uppercolumn), mouse (middle column) and human being (lower column) in whichthe base wherefrom an exon region of the full-length cPro Leu Ala 2starts is named 1.

[0027]FIG. 13 is a picture (as a substitute for a drawing) showing theresult of investigation of expression of KIDS cPLA 2 of the presentinvention using nerve stem cells and mature nerve cells.

[0028] In FIG. 13, the upper column is nestin as a control, the middlecolumn is the case where nerve stem cells were used and the lower columnis the case where mature cells of nerve were used. Pictures on the leftside (A) show the positions of each of the cells, pictures on the middle(B) are coloration showing the expression of KIDS cPLA 2 of the presentinvention and pictures at the right side are those where A at the leftside and B at the middle were piled to confirm the positions in both.

[0029]FIG. 14 is a picture (as a substitute for a drawing) showing theresult of investigation of expression of KIDS cPLA 2 of the presentinvention using nerve stem cells by means of stimulation with kainicacid, with kainic acid and CNQX and with glutamic acid. In FIG. 14, theprobe used is P90-P27 of 252 bp in the upper column, P19-P27 of 290 bpin the second column and G3 PDH and nestin in the lower two columns ascontrols. The lowermost picture of FIG. 14 shows the initiationpositions for transcription of KIDS cPLA 2 at the 5′-side and sequentialpositions of the probes used in the upper two columns in FIG. 14. Thelanes in FIG. 14 are control and stimulations by kainic acid (KA (10μM)), by kainic acid and CNQX (KA (10 μM)+CNQX (20 μM)) and by glutamicacid (Glu (50 μM)) from the left side.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] The present invention relates to a calcium-independent novelphospholipase A2 and, more particularly, it relates to acalcium-independent and hippocampus-specific phospholipase A2 which is aphospholipase A2 having an amino acid sequence described in SEQ ID NO:1, NO: 5 or NO: 8 of the Sequence Listing or an amino acid sequencewhere one or more amino acid(s) in the amino acid sequence is/aresubstituted with other amino acid(s) or deficient or one or more aminoacid(s) is/are added thereto, to gene coding therefor and an antibodywhere full length or fragment thereof is an antigen.

[0031] The present further relates to gene having a base sequenceexisting in intron where the base sequence is able to make theinitiation of transcription of RNA by external stimulation such askainic acid stimulation or electric stimulation possible and, moreparticularly, it relates to gene which is able to make the initiation oftranscription of RNA possible in a site-specific manner. Preferredexamples of the gene of the present invention are genes having the basesequence described in SEQ ID NO: 12, NO: 13 or NO: 14 of the SequenceListing and having the base sequence comprising a partial sequence wherea part thereof is deleted, added or substituted.

[0032] The present invention further relates to a promoter having a basesequence existing in intron and being able to make the initiation oftranscription of RNA by external stimulation such as kainic acidstimulation or electric stimulation possible and, more particularly, itrelates to the above-mentioned promoter where the initiation oftranscription of RNA is in a site-specific manner and to a regulatorygene having a regulatory element at the upper stream of the saidpromoter.

[0033] The present invention furthermore relates to a process whereinany of the above-mentioned gene, the above-mentioned promoter or theabove-mentioned regulatory gene is introduced into the upper stream ofthe gene coding for protein to initiate the transcription of RNA byexternal stimulation such as kainic acid stimulation or electricstimulation preferably in a site-specific manner whereby the saidprotein is expressed in response to the external stimulation and to aliving thing wherein any of the above-mentioned gene, theabove-mentioned promoter and the above-mentioned regulatory gene isintroduced into the upper stream of the gene coding for protein.

[0034] The present invention still further relates to a method for aspecific investigation of nerve stem cells by expression of KIDS cPLA 2of the present invention. Thus, the present invention relates to amethod for the detection or the identification of nerve stem cells bydetecting or identifying the mRNA coding for a calcium-independent andnovel phospholipase A2 (to be more specific, a phospholipase A2 which iscalcium-independent and hippocampus-specific and has an amino acidsequence described in SEQ ID NO: 1, NO: 5 or NO: 8 of the SequenceListing or an amino acid sequence where one or more amino acid(s) is/aresubstituted with other amino acid or deleted or one or more aminoacid(s) is/are added) by stimulating the nerve cells by externalstimulation.

[0035] During a course of the study for investigating the function ofphospholipase A2 in the brain, the present inventors prepared slices ofthe brain of rat into which kainic acid was intraperitoneally injectedand histochemically checked the expression of mRNA using cPLA2 as aprobe (searching element). For the selection of a probe, confirmationwas carried out by means of a northern blotting usually using differentsites of the cPLA2 whereupon it was found that, when a specific site(5′-terminal) was used, mRNA having a shorter length (about 1.8 kilobase pairs) than cPLA2 was induced.

[0036] Result of the northern blotting is shown in FIG. 1 as a picturewhich is a substitute for a drawing. The upper column of FIG. 1 shows abase sequence of cPLA2. The left end is a translation initiation codon(ATG) and the parts used as a probe are shown by A, B, C and D. Thus,probe A is a part from BamHI to BalI, probe B is a part from RsaI toRsaIBalI, probe C is a part from RsaI to BalI and probe D is a part fromRsaI to termination codon (TGA).

[0037] The middle column of FIG. 1 is the case where no kainic acidtreatment was carried out (KA(−)). The lower column of FIG. 1 is thecase after 3 hours from the kainic acid treatment (KA(+), 3 h). When nokainic acid treatment was carried out (KA(−)) (the middle column of FIG.1), a plot was noted at the position of cPLA2α only while, in the caseof 3 hours after a kainic acid treatment when that treatment was carriedout (KA(+)), plots of shorter chain length were able to be observed inprobes B, C and D at the 5′-terminal side not only at the position ofcPLA2α but also at the position beneath that.

[0038] Then, a northern blotting was carried out with a lapse of timefor hippocampus and cerebellum. The result is shown by a picture in FIG.2 as a substitute for a drawing. The left side of FIG. 2 is forhippocampus while the right side thereof is for cerebellum. Each of themshows the blots after 0 hour, 0.5 hour, 3 hours, 8 hours, 14 hours and18 hours from the kainic acid treatment. At any place of FIG. 2, plotwas found at the position of cPLA2α while, only at the area after 3hours from the kainic acid treatment in the case of hippocampus (leftside of FIG. 2), there were observed plots of shorter chain length notonly at the position of cPLA2α but also beneath that.

[0039] When an in situ hybridization was further carried out, a specificexpression was noted at dentate gyrus of hippocampus. The result isshown by a picture in FIG. 3 as a substitute for a drawing. The leftside of FIG. 3 is the result of a cross section of the brain while theright side thereof is that from a vertical section of the brain. In FIG.3, the part which is in black is a colored part. The colored part isdentate gyrus of hippocampus.

[0040] The result is enlarged and is shown in FIG. 4 which is a pictureas a substitute for a drawing. The left side of FIG. 4 is the resultwhere no kainic acid treatment was carried out while the right sidethereof is that after 3 hours from the kainic acid treatment. Colorationwas able to be observed around the dentate gyrus of hippocampus. Thiscoloration was strong at the outside of the dentate gyrus of hippocampusand, in the dentate gyrus of hippocampus, there are many nerve stemcells whereby that is presumed to be due to the nerve stem cellsexisting in the dentate gurus of hippocampus.

[0041] Therefore, the full length of cPLA2 was used as a probe and thedesired cDNA was obtained from a library of dentate gyrus ofhippocampus. This cDNA was translated into protein and its enzymaticactivity was checked whereupon a phospholipase A2 activity was found.

[0042] From the structure analysis, that was found to be a phospholipaseA2 molecule of a shortened type of phospholipase A2 of a cytoplasmictype (cytosolic phospholipase A2; abbreviated as cPLA2). cDNA of rat wasa protein with a molecular weight of 50,810.6 comprising 445 amino acidshaving 1,842 base pairs where the translated region was 1,355 basepairs. Since this phospholipase A2 of a shortened type is specificallyexpressed in the dentate gyrus of hippocampus after stimulation withkainic acid, it was named a kainate-inducible dentate gyrus specificcPLA2 (KIDS cPLA2).

[0043] Amino acid sequence of the resulting KIDS cPLA2 is shown by wayof one-letter code of amino acid as follows.

[0044] KIDS cPLA2 of human being is as follows. M N T T L S S L K E K VN T A Q C P L P 2 0 L F T C L H V K P D V S E L M F A D W V 4 0 E F S PY E I G M A K Y G T F M A P D L 6 0 F G S K F F M G T V V K K Y E E N PL H 8 0 F L M G V W G S A F S I L F N R V L G V 1 0 0 S G S Q S R G S TM E E E L E N I T T K 1 2 0 H I V S N D S S D S D D E S H E P K G T 1 40 E N E D A G S D Y Q S D N Q A S W I H R 1 6 0 M I M A L V S D S A L FN T R E G R A G 1 8 0 K V H N F M L G L N L N T S Y P L S P L 2 0 0 S DF A T Q D S F D D D E L D A A V A D 2 2 0 P D E F E R I Y E P L D V K SK K I H V 2 4 0 V D S G L T F N L P Y P L I L R P Q R G 2 6 0 V D L I IS F D F S A R P S D S S P P F 2 8 0 K E L L L A E K W A K M N K L P F PK I 3 0 0 D P Y V F D R E G L K E C Y V F K P K N 3 2 0 P D M B K D C PT I I H F V L A N I N F 3 4 0 R K Y K A P G V P R E T E E E K E I A D 36 0 F D I F D D P E S P F S T F N F Q Y P N 3 8 0 Q A F K R L H D L M HF N T L N N I D V 4 0 0 I K E A M V E S I E Y R R Q N P S R C S 4 2 0 VS L S N V E A R R F F N K E F L S K P 4 4 0 K A 4 4 2

[0045] KIDS cPLA2 of rat is as follows. M S T T L S S L K E K V S A A RG P L P 2 0 L F T C L H V K P D V S E L M F A D W V 4 0 E F S P Y E I GM A K Y G T F M T P D L 6 0 F G S K F F M G T V V K K Y E E N P L H 8 0F L M G V W G S A F S I L F N R V L G V 1 0 0 S G S Q N K G S T M E E EL E N I T A K 1 2 0 H I V S N D S S D S D D E A Q G P K G T 1 4 0 E N ED A E R E Y Q N D N Q A S W V H R 1 6 0 M L M A L V S D S A L F N T R BG R A G 1 8 0 K E H N F M L G L N L N T S Y P L S P L 2 0 0 R D F S P QD S F D D D E L D A A V A D 2 2 0 P D E F E R I Y E P L D V K S K K I HV 2 4 0 V D S G L T F N L P Y P L I L R P Q R G 2 6 0 V D L I I S F D FS A R P S D T S P P F 2 8 0 K E L L L A E K W A K M N K L P F P K I 3 00 D P Y V F D R E G L K E C Y V F K P K N 3 2 0 P D V E K D C P T I I HF V L A N I N F 3 4 0 R K Y K A P G V L R E T K E E K E I A D 3 6 0 F DI F D D P E S P F S T F N F Q Y P N 3 8 0 Q A F K R L H D L M Y F N T LN N I D V 4 0 0 I K D A I V E S I E Y R R Q N P S R C S 4 2 0 V S L S NV E A R K F F N K E F L S K P 4 4 0 T A E S I 4 4 5

[0046] KIDS cPLA2 of mouse is as follows. M S M T L S S L K E K V N A AR C P L P 2 0 L F T C L H V K P D V S E L M F A D W 4 0 V E F S P Y E IG M A K Y G T F M A P D 6 0 L F G S K F F M G T V V K K Y E E N P L 8 0H F L M G V W G S A F S I L F N R V L G 1 0 0 V S G S Q N K G S T M E EE L E N I T A 1 2 0 K H I V S N D S S D S D D E A Q G P K G 1 4 0 T E NE E A E K E Y Q S D N Q A S W V H 1 6 0 R M L M A L V S D S A L F N T RE G R A 1 8 0 G K V H N F M L G L N L N T S Y P L S P 2 0 0 L R D F S SQ D S F D D E L D A A V A D 2 2 0 P D E F E R I Y E P L D V K S K K I HV 2 4 0 V D S G L T F N L P Y P L I L R P Q R G 2 6 0 V D L I I S F D FS A R P S D T S P P F 2 8 0 K E L L L A E K W A K M N K L P F P K I 3 00 D P Y V F D R E G L K E C Y V F K P K N 3 2 0 P D V E K D C P T I I HF V L A N I N F 3 4 0 R K Y K A P G V L R E T K E E K E I A D 3 6 0 E DI F D D P E S P F S T F N F Q Y P N 3 8 0 Q A F K R L H D L M Y F N T LN N I D V 4 0 0 I K D A I V E S I E Y R R Q N P S R C S 4 2 0 V S L S NV E A R K F F N K E F L S K P 4 4 0 T V 4 4 2

[0047] Amino acid sequence of KIDS cPLA2 of human being is shown in SEQID NO: 1 of the Sequence Listing. Base sequence of translated region ofcDNA of KIDS cPLA2 of human being is shown in SEQ ID NO: 2, NO: 3 andNO: 4 of the Sequence Listing. SEQ ID NO: 2 is that where the sequenceof 5′ UTR is made type I, ID SEQ NO: 3 is that where the sequence of 5′UTR is made type II and SEQ ID NO: 4 is that where the sequence of 5′UTR is not classified into type I and type II.

[0048] Amino acid sequence of KIDS cPLA2 of rat is shown in SEQ ID NO: 5of the Sequence Listing. Base sequence of the translated region of cDNAof KIDS cPLA2 of rat is shown in SEQ ID NO: 6 and NO: 7 of the SequenceListing. SEQ ID NO: 6 is that for type I and SEQ ID NO: 7 is that fortype II.

[0049] Amino acid sequence of KIDS cPLA2 of mouse is shown in SEQ ID NO:8 of the Sequence Listing. Base sequence of the translated region ofcDNA of KIDS cPLA2 of mouse is shown in SEQ ID NO: 9, NO: 10 and NO: 11of the Sequence Listing. SEQ ID NO: 9 is that where the sequence of 5′UTR is made type I, ID SEQ NO: 10 is that where the sequence of 5′ UTRis made type II and SEQ ID NO: 11 is that where the sequence of 5′ UTRis not classified into type I and type II.

[0050] A polyclone antibody (stump antibody) which specificallyrecognizes the KIDS cPLA2 of the present invention was prepared.Expression of the desired protein was confirmed by immunohistochemicalanalysis using that antibody. The result is shown in FIG. 5 which is apicture as a substitute for a drawing. The left side of the upper columnof FIG. 5 is the case where no kainic acid treatment was carried outwhile the right side thereof is the case of 3 hours after the kainicacid treatment. Colored parts due to the antibody can be observed. Theleft side of the lower column of FIG. 5 is the control where notreatment with an anti-KIDS cPLA2 antibody (IgG) was carried out. Theright side of the lower column of FIG. 5 is the result of chromatographyin the absence of an anti-KIDS cPLA2 antibody (IgG) after 3 hours fromthe kainic acid treatment ((−) at the left side of the right side, lowercolumn, FIG. 5) and in the presence of that ((+) at the right sidethereof).

[0051] Then, cDNA coding for KIDS cPLA2 of the present invention andcPLA2 was integrated with an expression vector pTracer EF and itsexpression was investigated. The result is shown in FIG. 6 which is apicture as a substitute for a drawing. In FIG. 6, the lane 1 is the caseof a control vector, the lane 2 is the case of cPLA2α/pTracer EF and thelane 3 is the case of KIDS cPLA2/pTracer EF. In FIG. 6, the left side isthe case where an anti-V5 epitope IgG was used, the middle is the casewhere an anti-cPLA2α IgY was used and the right side is the case wherean anti-KIDS cPLA2 IgG was used.

[0052] Each spot by the anti-V5 epitope IgG and spot of KIDS cPLA2 bythe anti-KIDS cPLA2 IgG were confirmed whereby expression of KIDS cPLA2was confirmed.

[0053] Then enzymatic activity of cPLA2α and KIDS cPLA2 of the presentinvention were investigated. There were used 1-Pam-2-[¹⁴C]arachidonoyl-PC (black dots () in FIG. 7), 1-Pam-2-[¹⁴C]linoleoyl-PC(black triangles (▾) in FIG. 7), 1-Pam-2-[¹⁴C]oleoyl-PC (black squares(▪) in FIG. 7) and 1-Pam-2-[¹⁴C]palmitoyl-PC (asterisks (*) in FIG. 7)as the substrates for testing the enzymatic activity of each of them.The result is shown in FIG. 7. In FIG. 7, the left side is that for KIDScPLA2 of the present invention while the right side is that for cPLA2α.Each and any of the enzymes showed very high enzymatic activity toarachidonic acid phospholipids and was found to have nearly the sameactivity as phospholipase A2.

[0054] Values of those enzymatic activities (pmol/minute) are shown inthe following Table 1. TABLE 1 Enzymatic Activities of KIDS cPLA2 andcPLA2α Phospholipase A2 Activity (pmol/min) Substrate KIDS cPLA2 cPLA2α1-Pam-2-[¹⁴C]arachidonoyl-PC 35.6 ± 3.8 24.4 ± 1.41-Pam-2-[¹⁴C]linoleoyl-PC 20.1 ± 1.7 11.9 ± 1.8 1-Pam-2-[¹⁴C]oleoyl-PC14.3 ± 1.5  9.1 ± 1.1 1-Pam-2-[¹⁴C]palmitoyl-PC  9.4 ± 1.0  9.8 ± 1.7

[0055] Incidentally, the phospholipase A2 activity is given in terms ofthe difference from the control.

[0056] Then calcium dependency of cPLA2α and KIDS cPLA2 of the presentinvention on enzymatic activity was investigated using1-Pam-2-[¹⁴C]arachidonoyl-PC as a substrate.

[0057] The result is shown in FIG. 8 and FIG. 9. The solid lines in FIG.8 are the case of KIDS cPLA2 of the present invention while the brokenlines therein are the case of cPLA2α. In each of them, the black dots() are the data in the absence of EDTA while the open circles (◯) arethose in the presence of EDTA. It is noted that, in the case of cPLA2α,there is a sudden reduction in the activity in the absence of calciumdue to EDTA while, in the case of KIDS cPLA2 of the present invention,there is no such a reduction in the activity.

[0058]FIG. 9 shows the above-mentioned result in terms of a relativeratio. It is noted that, in the case of KIDS cPLA2 of the presentinvention, an activity of around 40% is maintained even in the absenceof calcium while, in the case of cPLA2α, the activity is reduced to anextent of around 10-15% in the absence of calcium.

[0059] As such, KIDS cPLA2 of the present invention is characteristic inbeing calcium-independent as compared with the conventional cPLA2α.

[0060] Then expression of KIDS cPLA2 of the present invention incPLA-defective mouse prepared by Shimizu, et al. (Uozumi, N. et al.,Nature, 390, 618-622, 1997) was investigated. The result is shown inFIG. 10 which is a picture as a substitute for a drawing. In FIG. 10,the upper column is that for (+/+) of a knockout mouse while the lowercolumn is that for (−/−) of a knockout mouse. In FIG. 10, the left sideis that where no kainic acid treatment was carried out (KA (−)) whilethe right side is that after 3 hours from the kainic acid treatment (KA(+)). In any of the knockout mice, expression of the present enzyme wasable to be confirmed by a kainic acid treatment.

[0061] The above shows that KIDS cPLA2 of the present inventionexpresses using a promoter which is different from its full-lengthcPLA2.

[0062]FIG. 11 illustrates the state of expression of cPLA2 and KIDScPLA2. In FIG. 11, the upper column schematically shows exon and intronof cPLA2 in genomic gene. The full-length cPLA2 is produced from allexons and regulatory gene containing a promoter region is present in theupper stream of the initial exon. On the contrary, KIDS cPLA2 of thepresent invention is a protein starting from the 308th methioninementioned as “M-308” in FIG. 11 and, since expression of this proteinwas confirmed in the cPLA-defective mouse, i.e., a mouse where thefunction of regulatory gene containing a promoter region in the upperstream of the initial exon, it has been found that KIDS cPLA2 of thepresent invention has a regulatory gene region containing a promoterregion in the upper stream of “M-308”. However, the said regulatory geneis in such a manner that, under an ordinary state, the gene does notfunction and has also been noted to function only by the stimulationsuch as by a kainic acid stimulation.

[0063] Therefore, a base sequence of upper stream of “M-308” wasanalyzed for rat, mouse and human being. The result is aligned and shownin FIG. 12.

[0064] The base sequence of this intron in human being is shown in SEQID NO: 7 of the Sequence Listing. The base sequence thereof in rat isshown in SEQ ID NO: 8 of the Sequence Listing. Further, the basesequence thereof in mouse is shown in SEQ ID NO: 9 of the SequenceListing.

[0065]FIG. 12 shows a numbering for base sequences from the first baseof intron immediately before exon containing “M-308” of rat (uppercolumn), mouse (middle column) and human being (lower column) in whichthe base wherefrom an exon region of the full-length cPLA2 starts isnamed No. 1. ATG from the 92nd one (human being) in this numbering is atranslation initiation codon of KIDS cPLA2.

[0066] Then, expression of KIDS cPLA 2 of the present invention wasinvestigated using nerve cells in the dentate gyrus of hippocampus ofthe brain. The result is shown in FIG. 13 which is a picture as asubstitute for a drawing.

[0067] In FIG. 13, the upper column is nestin as a control, the middlecolumn is the case where nerve stem cells were used and the lower columnis the case where mature cells of nerve were used. A in the left sideshows the positions of each of the cells, B in the middle is colorationshowing the expression of KIDS cPLA 2 of the present invention and theright side is that where A at the left side and B at the middle werepiled to confirm the positions in both.

[0068] As the result, it is noted that no clear expression is observedfor KIDS cPLA2 of the present invention in nerve mature cells but aclear expression is observed in nerve stem cells. The above suggeststhat KIDS cPLA2 of the present invention is a substance which isspecifically expressed in nerve stem cells and that, in nerve stemcells, intron in mature cells specifically plays a role of a promoter.

[0069] Then expression of KIDS cPLA 2 of the present invention usingnerve stem cells by means of stimulation by kainic acid (10 μM), bykainic acid and CNQX (10 μM KA and 20 μM CNQX) and by glutamic acid (50μM) was investigated.

[0070] The result is shown in FIG. 14 which is a picture as a substitutefor a drawing. In FIG. 14, the probe used is P90-P27 of 252 bp (the saidsequence is a sequence of a moiety which is common in the full-lengthcPLA2) in the upper column, P19-P27 of 290 bp (the said seauencecontains a sequence specific to KIDS cPLA2 of the present invention) inthe second column and G3 PDH and nestin in the lower two columns ascontrols. The lowermost picture of FIG. 14 shows the initiationpositions for transcription of KIDS cPLA 2 at the 5′-side and sequentialpositions of the probes used in the upper two columns in FIG. 14.

[0071] The lanes in FIG. 14 are control and stimulations by kainic acid(KA (10 μM)), by kainic acid and CNQX (KA (10 μM)+CNQX (20 μM)) and byglutamic acid (Glu (50 μM)) from the left side.

[0072] As a result, in the case of stimulation by kainic acid (10 μM), aspecific expression of KIDS cPLA2 of the present invention wasconfirmed.

[0073] Accordingly, the present invention provides a method forsearching the nerve stem cells in a specific manner by expression ofKIDS cPLA2 of the present invention. Thus, according to such a method ofthe present invention, cells which are to be the candidates arestimulated by kainic acid and expression of KIDS cPLA2 of the presentinvention is observed whereby the nerve stem cells are able to bespecifically and easily searched.

[0074] The KIDS cPLA2 of the present invention is in a partial length ofthe full-length cPLA2. It is characterized in maintaining aphospholipase AZ activity and having a calcium-independent property andis not always limited to that having an amino acid sequence described inSEQ ID NO: 1, NO: 3 or NO: 5 of the Sequence Listing. So far as itmaintains the phospholipase A2 activity and is calcium-independent,about 1-200 or preferably about 1-100, 1-50 or 1-20 amino acid(s)described in SEQ ID NO: 1, NO: 3 or NO: 5 of the Sequence Listing may besubstituted with other amino acid(s) or may be deleted therefrom oradded thereto. It is also possible that such substitution, deletion andaddition may be simultaneously carried out in a combined manner.

[0075] Although the KIDS cPLA2 of the present invention may bemanufactured according to a process disclosed in the presentspecification, it may also be manufactured by a conventional generecombination technique using cDNA of the KIDS cPLA2 of the presentinvention.

[0076] When a full length or a part of the KIDS cPLA2 of the presentinvention or, preferably, a peptide comprising 10 or more amino acids isused as an antigen, it is possible to manufacture an antibody thereto.The antibody of the present invention may be manufactured by aconventional process and, if necessary, it is possible to manufacture apolyclonal antibody or a monoclonal antibody.

[0077] It has been also known that cell death specific to the dentategyrus of hippocampus takes place by a kainic acid stimulation, by a fitof epilepsy, etc. The present inventors have found that KIDS cPLA2 isexpressed in the dentate gyrus of hippocampus by a kainic acidstimulation, by a fit of epilepsy, etc. In view of the above, cell deathin the dentate gyrus of hippocampus can be prevented by preparing aninhibitor for the said enzyme and the said enzyme is useful for adevelopment of such an inhibitor as well.

[0078] Further, the present inventors have found for the first time thatsome of intron has a function as a regulatory gene which is activated byan external stimulation and have clarified that at least a base sequencewhich also functions as a promoter responding to the externalstimulation is present in the base sequence of intron.

[0079] Accordingly, the present invention provides a gene having a basesequence existing in intron where the said base sequence is able to makethe initiation of transcription of RNA by external stimulation possible.The said gene of the present invention comprises at least six bases.Preferably, it is an oligonucleotide having a base sequence existing inintron and comprising at least four or, preferably, at least six basesin the base sequence shown in SEQ ID NO: 7, NO: 8 or NO: 9 of theSequence Listing where the said base sequence is able to make theinitiation of transcription of RNA by external stimulation possible.

[0080] Since the said gene of the present invention has a function atleast as a promoter participating in initiation of transcription of RNA,the present invention also provides a promoter comprising the said geneor its partial length and being able to make the initiation oftranscription of RNA by external stimulation possible. The promoter ofthe present invention is characterized in that it does not generate theinitiation of transcription of RNA under an ordinary condition of maturecells but is able to generate the initiation of transcription of RNAonly by a specific external stimulation. The promoter of the presentinvention is also characterized in that its base sequence is a basesequence existing in intron. More preferably, it is characterized inthat the site whereby the initiation of transcription of RNA is madepossible is specific. It is preferred that the promoter of the presentinvention has a length of at least 4-20 bases or, preferably, at least6-20 bases although the present invention is not limited thereto.

[0081] Although the promoter of the present invention may be usedsolely, it is preferred to use it together with a regulatory elementsuch as an enhancer. Although the regulatory enhancer is positioned atcis, it may be at trans as well. The present invention provides aregulatory gene in which the regulatory element and the promoter of thepresent invention as mentioned above are in a set. When the regulatoryelement is a cis-element, such a regulatory gene may be insingle-stranded or may be in double-stranded. It is used as doublestrands when the regulatory element is a trans-element.

[0082] When an intron is known to have a base sequence which makes theinitiation of transcription of RNA by external stimulation possiblewhile it is not well known that which base sequence in the intron playsa role as a promoter, etc., then the full length of the said intron maybe used as a regulatory gene of the present invention.

[0083] The term “external stimulation” used in the present invention isa stimulation which does not take place under the growth condition ofordinary mature cells and, preferably, it is a stimulation by which celldeath is induced. Thus, there may be exemplified stimulation by achemical substance such as kainic acid; physical stimulation such aselectric shock, temperature change, etc.; stimulation by disorder ofother organs such as a fit of epilepsy; and the like.

[0084] The term “site-specific” used in the present invention means thata thing is specific to a site which is able to be discriminated fromothers in terms of type, state, growth degree, etc. of tissues, organsor cells in living body. Although the promoter, gene, etc. of thepresent invention which are able to make the initiation of transcriptionof RNA by external stimulation possible may not be always site-specific,they may be site-specific as well. The base sequence of intron shown bySEQ ID NO: 7, NO: 8 and NO: 9 of the Sequence Listing of the presentspecification is believed to be specific to the dentate gyrus ofhippocampus although the promoter, gene, etc. of the present inventionis not limited thereto.

[0085] The present invention is to clarify the presence of a basesequence which makes the initiation of transcription of RNA by externalstimulation in intron of living thing possible and the range ofutilization of such a gene of the present invention is quite broad. Thefirst characteristic is that, since it is present as intron, even whenthe gene is introduced, it usually functions only as intron and does notaffect the ordinary growth of living things. The second characteristicis that the regulatory gene of the present invention is inactive for thetranscription of RNA under an ordinary state and does not express theprotein coded at its downstream. The third characteristic is that it isalso possible to make it expressed in a site-specific manner.

[0086] Since the promoter and regulatory gene of the present inventionhave such characteristics, applications according to the particularobject are possible. For example, when it is an object that a partiallength of a protein is expressed whereby its physiological activity isobserved in vivo, the gene of the present invention is introducedimmediately before the exon containing methionine which is to be aninitiation codon to give an external stimulation to living body wherebyexpression of protein having a desired partial length can be promoted.When there is no suitable methionine, it may be also possible tointroduce a base sequence coding for methionine into an intron region.

[0087] According to the second characteristic, gene bonded with theregulatory gene and promoter of the present invention at the upperstream of the desired protein is introduced into living body to give aspecific external stimulation whereby the expression of the desiredintroduced protein is expressed only at the stage of giving the externalstimulation. For example, it is possible that a physiologically activeprotein is added to the end of the promoter of the present inventionand, only when a specific external stimulation is applied, the saidphysiologically active protein is expressed and the said physiologicalactivity is temporarily given to the cell. When a toxin such asdiphtheria toxin is used as a physiologically active protein, it is nowpossible to kill the cells in a transient manner. Alternatively, thegene to which CRE gene is connected to the downstream of the promoter ofthe present invention is introduced whereupon there is prepared atransgenic mouse where specific gene such as glutamic acid receptor issurrounded by a lox-P sequence. By doing so, CRE gene is expressed andspecific gene such as glutamic acid receptor surrounded by a lox-Psequence is deleted by a homologous recombination when a specificexternal stimulation is applied and, therefore, it is possible toprepare a mouse deficient in specific gene such as a glutamic acidreceptor as from the stage of application of a specific externalstimulation. It is now possible by such a transgenic mouse to preciselyanalyze the pathology in mature living body where specific gene such asa glutamic acid receptor is deficient.

[0088] Further, according to the above-mentioned third characteristic,it is possible to bring about the above-mentioned characteristic in asite-specific manner in a living body. For example, it is possible todestroy a specific gene specifically in the dentate gyrus ofhippocampus.

[0089] Accordingly, the present invention provides a method whereexpression of gene coding for a protein introduced into a living bodyusing the promoter and regulatory gene of the present invention isregulated by a specific external stimulation. As mentioned already, itis possible according to this method of the present invention thatexpression length, expression time and expression site of the introducedprotein are regulated.

[0090] There is no particular limitation as to the protein which isintroduced in this method of the present invention provided that it is aprotein having any physiological activity and the protein can beintroduced in a state of genome or in a state of cDNA. The proteinhaving a physiological activity may be, for example, that which has theso-called physiological activity such as hormones and cytokines, toxinsuch as diphtheria toxin or that which induces a homologousrecombination such as CRE gene.

[0091] The present invention also provides a living thing into which thepromoter and regulatory gene of the present invention are introduced atthe upper stream of the gene coding for protein. The living thing of thepresent invention is useful as an experimental animal and is applicable,for example, to mouse, rat, rabbit and monkey. It is also possible toapply to plants.

[0092] With regard to such experimental animals, there have beendeveloped transgenic mouse, knockout mouse, etc. In a knockout mouse,there has been a demand for development of a conditional targetingmethod and, as being noted from the fact that a tissue-specificallyexpressing promoter, a tetracycline-sensitive promoter, etc. have beendeveloped, there has been a demand for development of a promoter whichis tissue-specific and stage-specific. The promoter and regulatory geneof the present invention satisfy such requirements and also have afunction as intron and, therefore, the promoter and regulatory gene ofthe present invention can be widely applied to experimental animals.

EXAMPLES

[0093] Now the present invention will be illustrated in more detail byway of the following Examples although the present invention is notlimited to those Examples only.

Example 1 Northern Blotting Using Various Probes of cPLA2

[0094] cDNA of cPLA2a of rat was divided into four main region, i.e., A,B, C and D from the 5′-terminal. Length of each region was made around300-500 bp and, after such a cDNA fragment was integrated with ariboprobe synthetic vector, a radio-labeled riboprobe was synthesized byan in vitro transcription method. A hybridization reaction was carriedout using a membrane which was blotted with poly(A)⁺RNA of hippocampaldentate gyrus and hippocampus of rat subjected to a kainic acidstimulation and a riboprobe of each of A, B, C and D to check whichprobe was able to detect the KIDS cPLA2. As a result, it was found thatKIDS cPLA2 mRNA was detected in riboprobes of B, C and D except A.

[0095] The result is shown in FIG. 1 and FIG. 2.

Example 2 In Situ Hybridization

[0096] Brain of Wister rat of 3 weeks age stimulated by kainic acid wasfixed by 4% paraformaldehyde and then a frozen slice was prepared. Eachof the radio-labeled riboprobes B, C and D was subjected to ahybridization reaction with the frozen slice and the labeled image ofthe slice from each probe was confirmed to be same. Then, frozen sliceof the brain was prepared again using riboprobe C with or without akainic acid stimulation and an expression pattern of KIDS cPLA2 mRNA wasinvestigated. As a result, KIDS cPLA2 mRNA was found to be drasticallyinduced in the hippocampal dentate gyrus. When a strongly enlarged imagewas observed under a microscope, KIDS cPLA2 mRNA was found to expressparticularly abundantly in the innermost layer of the dentate gyrus.

[0097] The result is shown in FIG. 3 and FIG. 4.

Example 3 Immunohistochemical Dyeing

[0098] Brains of cPLA2a knockout mouse and C57/Black 6J mouse of 6-10weeks age and Wister rat of 3 weeks age stimulated by kainic acid werefixed with 4% paraformaldehyde and then frozen slices were prepared.Immunoreaction of an anti-KIDS cPLA2-specific stump antibody with thebrain slice was carried out overnight at 4° C. and expression of KIDScPLA2 protein was confirmed by a secondary antibody labeled with goldcolloid. As a result, it was found that, like mRNA, KIDS cPLA2 wasdrastically induced in the hippocampal dentate gyrus and further thatsuch an expression was noted in the hippocampal dentate gyrus of cPLA2aknockout mouse as well. From the above, it was suggested that a promoterof KIDS cPLA2 was present at the downstream of the eighth exon of cPLA2adestroyed by a cPLA2a knockout mouse and found that an isoform of cPLA2awas induced by acute nervous stimulation.

[0099] The result is shown in FIG. 5.

Example 4 Cloning of cDNA of KIDS cPLA2 of Rat

[0100] Clone was isolated after confirming its presence by two kinds ofmethod.

[0101] (1) A cDNA library was prepared using poly(A)⁺RNA purified fromhippocampus of rat after stimulation by kainic acid and a positive clonewas selected using a cDNA sequence of 1,365 (Rsa I)-1,925 (Bal I) fromthe initiation point for translation of cPLA2a which is able to detectKIDS cPLA2 as a probe.

[0102] From 4,000,000 clones were selected 12 positive clones. Amongthem, two are those of full-length phospholipase A2 while six and fouramong the residual ten were different it their type. The former wasnamed type II and the latter was named type I.

[0103] (2) In order to confirm the 5′-terminal of KIDS cPLA2 cDNA ofrat, a 5′ RACE method (5′-rapid amplification of cDNA ends) was carriedout using poly(A)⁺RNA purified from the hippocampus of rat afterstimulation by kainic acid. A sequence amplified to the 5′-upper streamfrom the primer existing in the above-mentioned sequence of 1,365 (RsaI)-1,925 (Bal I) was identical with a sequence of clone selected fromthe cDNA library.

[0104] From the above, KIDS cPLA2 was found to be a novel gene inducedin hippocampus after stimulation by kainic acid.

[0105] An amino acid sequence of the resulting KIDS cPLA2 of rat isshown in SEQ ID NO: 5 of the Sequence Listing. Base sequences oftranslated region of cDNA of KIDS cPLA2 of rat are shown in SEQ ID NO: 6(type I) and SEQ ID NO: 7 (type II) of the Sequence Listing.

[0106] An amino acid sequence of KIDS cPLA2 of mouse is shown in SEQ IDNO: 8 of the Sequence Listing. Base sequences of translated region ofKIDS cPLA2 of mouse are shown in SEQ ID NO: 9 (in the case that 5′UTR isdefined as type I), in SEQ ID NO: 10 (in the case that 5′UTR is definedas type II) and in SEQ ID NO: 11 (in the case that 5′UTR is not dividedinto types I and II), respectively, of the Sequence Listing.

[0107] An amino acid sequence of KIDS cPLA2 of human being is shown inSEQ ID NO: 1 of the Sequence Listing. Base sequences of translatedregion of cDNA of KIDS cPLA2 of human being are shown in SEQ ID NO: 2(when 5′UTR was made type I), SEQ ID NO: 3 (when 5′UTR was made type II)and SEQ ID NO: 4 (when 5′UTR was not divided into types I and II)of theSequence Listing.

[0108] Those sequences are selected from the most appropriate sequencesafter the cDNA sequences of human being, mouse and rat were subjected toan alignment program at the same time and then applied with theconditions such as the position of initiation of transcription, theposition of nucleotide presumed to be the transcription initiationposition for each of type I and type II, the junction sequenceconnecting the sequence of type I and the sequence of type II, thesequence homology as a whole, etc.

Example 5 Manufacture of an Antibody of KIDS cPLA2

[0109] In order to specifically detect the KIDS cPLA2 having theentirely same sequence from a sequence of Met-308 of cPLA2a of rat, asynthetic peptide having an amino acid sequence comprising 7 startingfrom this Met-308 (MSTTLSS) was immunized to rabbit and its serumfraction was prepared. Further, immunoglobulin (IgG) was purified fromthis fraction to give a final specimen. Incidentally, this stumpantibody was confirmed to specifically recognize not only KIDS cPLA2 ofrat but also KIDS cPLA2 of mouse.

Example 6 Analysis of Base Sequence of Intron

[0110] Analysis of intron sequences (assumed promoter region) of KIDScPLA2 of rat and mouse was carried out by the following method.

[0111] It was investigated whether a fundamental transcription activitywas present for a region ranging up to about 9 kb upstream including 5′UTR of KIDS cPLA2 of rat and mouse (a region until exon of cPLAadestroyed in knockout mouse). Firstly, there was constructed a reportervector where each of a sequence of about 9 kb of this region, a sequenceincluding about 1,000 bp upstream of 5′ UTR, a sequence including about500 bp having a high homology among human being, rat and mouse and asequence of about 700 bp including 5′UTR was integrated with the upperstream of luciferase gene. Such a reporter vector was introduced into anincubated cell strain, the supernatant liquid of the cells was preparedand its luciferase activity was measured as an index for a fundamentaltranscription activity. As a result, a sequence of about 700 bpincluding 5′ UTR was found to have an especially high transcriptionactivity.

[0112] The result is shown in FIG. 12. Further, base sequences of theanimals are shown in SEQ ID NO: 12 (human being), SEQ ID NO: 13 (rat)and SEQ ID NO: 14 (mouse).

INDUSTRIAL APPLICABILITY

[0113] The present invention provides a novel enzyme which is presumedto be a case for cell death specific to the hippocampal dentate gyrus instimulation by kainic acid, fit of epilepsy, etc. It is now possible toprevent the cell death by preparing an inhibitor of this enzyme.

[0114] The present invention also provides a novel enzyme having aphospholipase A2 activity and being calcium-independent.

[0115] The present invention further clarifies for the first time that,in intron, there is a function of making the initiation of transcriptionof RNA by an external stimulation possible. A novel function of intronin genome is elucidated and, at the same time, there is provided a newtype of gene which functions as a promoter or as a regulatory gene by anexternal stimulation. The new type of gene functioning as a promoter oras a regulatory gene according to the present invention not only has afunction as intron but also makes the expression of a stage-specificdesired gene in response to an external stimulation possible and furthermakes the expression of the site-specific desired gene depending uponthe tissue possible. Consequently, the promoter or the regulatory geneof the present invention is able to be used for the regulation ofexpression of gene and is applicable to transgenic animals, knockoutanimals, etc.

1 14 1 442 PRT Human 1 Met Asn Thr Thr Leu Ser Ser Leu Lys Glu Lys ValAsn Thr Ala Gln 1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys Leu His ValLys Pro Asp Val Ser 20 25 30 Glu Leu Met Phe Ala Asp Trp Val Glu Phe SerPro Tyr Glu Ile Gly 35 40 45 Met Ala Lys Tyr Gly Thr Phe Met Ala Pro AspLeu Phe Gly Ser Lys 50 55 60 Phe Phe Met Gly Thr Val Val Lys Lys Tyr GluGlu Asn Pro Leu His 65 70 75 80 Phe Leu Met Gly Val Trp Gly Ser Ala PheSer Ile Leu Phe Asn Arg 85 90 95 Val Leu Gly Val Ser Gly Ser Gln Ser ArgGly Ser Thr Met Glu Glu 100 105 110 Glu Leu Glu Asn Ile Thr Thr Lys HisIle Val Ser Asn Asp Ser Ser 115 120 125 Asp Ser Asp Asp Glu Ser His GluPro Lys Gly Thr Glu Asn Glu Asp 130 135 140 Ala Gly Ser Asp Tyr Gln SerAsp Asn Gln Ala Ser Trp Ile His Arg 145 150 155 160 Met Ile Met Ala LeuVal Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu 165 170 175 Gly Arg Ala GlyLys Val His Asn Phe Met Leu Gly Leu Asn Leu Asn 180 185 190 Thr Ser TyrPro Leu Ser Pro Leu Ser Asp Phe Ala Thr Gln Asp Ser 195 200 205 Phe AspAsp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe 210 215 220 GluArg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val 225 230 235240 Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg 245250 255 Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg260 265 270 Pro Ser Asp Ser Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala GluLys 275 280 285 Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp ProTyr Val 290 295 300 Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe LysPro Lys Asn 305 310 315 320 Pro Asp Met Glu Lys Asp Cys Pro Thr Ile IleHis Phe Val Leu Ala 325 330 335 Asn Ile Asn Phe Arg Lys Tyr Lys Ala ProGly Val Pro Arg Glu Thr 340 345 350 Glu Glu Glu Lys Glu Ile Ala Asp PheAsp Ile Phe Asp Asp Pro Glu 355 360 365 Ser Pro Phe Ser Thr Phe Asn PheGln Tyr Pro Asn Gln Ala Phe Lys 370 375 380 Arg Leu His Asp Leu Met HisPhe Asn Thr Leu Asn Asn Ile Asp Val 385 390 395 400 Ile Lys Glu Ala MetVal Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro 405 410 415 Ser Arg Cys SerVal Ser Leu Ser Asn Val Glu Ala Arg Arg Phe Phe 420 425 430 Asn Lys GluPhe Leu Ser Lys Pro Lys Ala 435 440 2 1908 DNA Human 2 gattttgattggaagtacta ttttgaatag cattctttct gtgtctgttt ataaatttaa 60 agtcatctttttctttcttc tgtggacaga gaatgaatac tactctgagc agtttgaagg 120 aaaaagttaatactgcacaa tgccctttac ctcttttcac ctgtcttcat gtcaaacctg 180 acgtttcagagctgatgttt gcagattggg ttgaatttag tccatacgaa attggcatgg 240 ctaaatatggtacttttatg gctcccgact tatttggaag caaatttttt atgggaacag 300 tcgttaagaagtatgaagaa aaccccttgc atttcttaat gggtgtctgg ggcagtgcct 360 tttccatattgttcaacaga gttttgggcg tttctggttc acaaagcaga ggctccacaa 420 tggaggaagaattagaaaat attaccacaa agcatattgt gagtaatgat agctcggaca 480 gtgatgatgaatcacacgaa cccaaaggca ctgaaaatga agatgctgga agtgactatc 540 aaagtgataatcaagcaagt tggattcatc gtatgataat ggccttggtg agtgattcag 600 ctttattcaataccagagaa ggacgtgctg ggaaggtaca caacttcatg ctgggcttga 660 atctcaatacatcttatcca ctgtctcctt tgagtgactt tgccacacag gactcctttg 720 atgatgatgaactggatgca gctgtagcag atcctgatga atttgagcga atatatgagc 780 ctctggatgtcaaaagtaaa aagattcatg tagtggacag tgggctcaca tttaacctgc 840 cgtatcccttgatactgaga cctcagagag gggttgatct cataatctcc tttgactttt 900 ctgcaaggccaagtgactct agtcctccgt tcaaggaact tctacttgca gaaaagtggg 960 ctaaaatgaacaagctcccc tttccaaaga ttgatcctta tgtgtttgat cgggaagggc 1020 tgaaggagtgctatgtcttt aaacccaaga atcctgatat ggagaaagat tgcccaacca 1080 tcatccactttgttctggcc aacatcaact tcagaaagta caaggctcca ggtgttccaa 1140 gggaaactgaggaagagaaa gaaatcgctg actttgatat ttttgatgac ccagaatcac 1200 cattttcaaccttcaatttt caatatccaa atcaagcatt caaaagacta catgatctta 1260 tgcacttcaatactctgaac aacattgatg tgataaaaga agccatggtt gaaagcattg 1320 aatatagaagacagaatcca tctcgttgct ctgtttccct tagtaatgtt gaggcaagaa 1380 gatttttcaacaaggagttt ctaagtaaac ccaaagcata gttcatgtac tggaaatggc 1440 agcagtttctgatgctgagg cagtttgcaa tcccatgaca actggattta aaagtacagt 1500 acagatagtcgtactgatca tgagagactg gctgatactc aaagttgcag ttacttagct 1560 gcatgagaataatactatta taagttaggt gacaaatgat gttgattatg taaggatata 1620 cttagctacattttcagtca gtatgaactt cctgatacaa atgtagggat atatactgta 1680 tttttaaacatttctcacca actttcttat gtgtgttctt tttaaaaatt ttttttcttt 1740 taaaatatttaacagttcaa tctcaataag acctcgcatt atgtatgaat gttattcact 1800 gactagatttattcatacca tgagacaaca ctatttttat ttatatatgc atatatatac 1860 atacatgaaataaatacatc aatataaaaa taaaaaaaaa cggaattc 1908 3 1925 DNA Human 3gattattttt taaatgaaga tagttacttc catagagctt attttttgtt gttcattcag 60gacctagtaa tttctagaag taataagact tatttttatt ataaagagaa tgaatactac 120tctgagcagt ttgaaggaaa aagttaatac tgcacaatgc cctttacctc ttttcacctg 180tcttcatgtc aaacctgacg tttcagagct gatgtttgca gattgggttg aatttagtcc 240atacgaaatt ggcatggcta aatatggtac ttttatggct cccgacttat ttggaagcaa 300attttttatg ggaacagtcg ttaagaagta tgaagaaaac cccttgcatt tcttaatggg 360tgtctggggc agtgcctttt ccatattgtt caacagagtt ttgggcgttt ctggttcaca 420aagcagaggc tccacaatgg aggaagaatt agaaaatatt accacaaagc atattgtgag 480taatgatagc tcggacagtg atgatgaatc acacgaaccc aaaggcactg aaaatgaaga 540tgctggaagt gactatcaaa gtgataatca agcaagttgg attcatcgta tgataatggc 600cttggtgagt gattcagctt tattcaatac cagagaagga cgtgctggga aggtacacaa 660cttcatgctg ggcttgaatc tcaatacatc ttatccactg tctcctttga gtgactttgc 720cacacaggac tcctttgatg atgatgaact ggatgcagct gtagcagatc ctgatgaatt 780tgagcgaata tatgagcctc tggatgtcaa aagtaaaaag attcatgtag tggacagtgg 840gctcacattt aacctgccgt atcccttgat actgagacct cagagagggg ttgatctcat 900aatctccttt gacttttctg caaggccaag tgactctagt cctccgttca aggaacttct 960acttgcagaa aagtgggcta aaatgaacaa gctccccttt ccaaagattg atccttatgt 1020gtttgatcgg gaagggctga aggagtgcta tgtctttaaa cccaagaatc ctgatatgga 1080gaaagattgc ccaaccatca tccactttgt tctggccaac atcaacttca gaaagtacaa 1140ggctccaggt gttccaaggg aaactgagga agagaaagaa atcgctgact ttgatatttt 1200tgatgaccca gaatcaccat tttcaacctt caattttcaa tatccaaatc aagcattcaa 1260aagactacat gatcttatgc acttcaatac tctgaacaac attgatgtga taaaagaagc 1320catggttgaa agcattgaat atagaagaca gaatccatct cgttgctctg tttcccttag 1380taatgttgag gcaagaagat ttttcaacaa ggagtttcta agtaaaccca aagcatagtt 1440catgtactgg aaatggcagc agtttctgat gctgaggcag tttgcaatcc catgacaact 1500ggatttaaaa gtacagtaca gatagtcgta ctgatcatga gagactggct gatactcaaa 1560gttgcagtta cttagctgca tgagaataat actattataa gttaggtgac aaatgatgtt 1620gattatgtaa ggatatactt agctacattt tcagtcagta tgaacttcct gatacaaatg 1680tagggatata tactgtattt ttaaacattt ctcaccaact ttcttatgtg tgttcttttt 1740aaaaattttt tttcttttaa aatatttaac agttcaatct caataagacc tcgcattatg 1800tatgaatgtt attcactgac tagatttatt cataccatga gacaacacta tttttattta 1860tatatgcata tatatacata catgaaataa atacatcaat ataaaaataa aaaaaaacgg 1920aattc 1925 4 2020 DNA Human 4 gattattttt taaatgaaga tagttacttccatagagctt attttttgtt gttcattcag 60 gacctagtaa tttctagaag taataagacttatttttatt ataaagttat aagattttga 120 ttggaagtac tattttgaat agcattctttctgtgtctgt ttataaattt aaagtcatct 180 ttttctttct tctgtggaca gagaatgaatactactctga gcagtttgaa ggaaaaagtt 240 aatactgcac aatgcccttt acctcttttcacctgtcttc atgtcaaacc tgacgtttca 300 gagctgatgt ttgcagattg ggttgaatttagtccatacg aaattggcat ggctaaatat 360 ggtactttta tggctcccga cttatttggaagcaaatttt ttatgggaac agtcgttaag 420 aagtatgaag aaaacccctt gcatttcttaatgggtgtct ggggcagtgc cttttccata 480 ttgttcaaca gagttttggg cgtttctggttcacaaagca gaggctccac aatggaggaa 540 gaattagaaa atattaccac aaagcatattgtgagtaatg atagctcgga cagtgatgat 600 gaatcacacg aacccaaagg cactgaaaatgaagatgctg gaagtgacta tcaaagtgat 660 aatcaagcaa gttggattca tcgtatgataatggccttgg tgagtgattc agctttattc 720 aataccagag aaggacgtgc tgggaaggtacacaacttca tgctgggctt gaatctcaat 780 acatcttatc cactgtctcc tttgagtgactttgccacac aggactcctt tgatgatgat 840 gaactggatg cagctgtagc agatcctgatgaatttgagc gaatatatga gcctctggat 900 gtcaaaagta aaaagattca tgtagtggacagtgggctca catttaacct gccgtatccc 960 ttgatactga gacctcagag aggggttgatctcataatct cctttgactt ttctgcaagg 1020 ccaagtgact ctagtcctcc gttcaaggaacttctacttg cagaaaagtg ggctaaaatg 1080 aacaagctcc cctttccaaa gattgatccttatgtgtttg atcgggaagg gctgaaggag 1140 tgctatgtct ttaaacccaa gaatcctgatatggagaaag attgcccaac catcatccac 1200 tttgttctgg ccaacatcaa cttcagaaagtacaaggctc caggtgttcc aagggaaact 1260 gaggaagaga aagaaatcgc tgactttgatatttttgatg acccagaatc accattttca 1320 accttcaatt ttcaatatcc aaatcaagcattcaaaagac tacatgatct tatgcacttc 1380 aatactctga acaacattga tgtgataaaagaagccatgg ttgaaagcat tgaatataga 1440 agacagaatc catctcgttg ctctgtttcccttagtaatg ttgaggcaag aagatttttc 1500 aacaaggagt ttctaagtaa acccaaagcatagttcatgt actggaaatg gcagcagttt 1560 ctgatgctga ggcagtttgc aatcccatgacaactggatt taaaagtaca gtacagatag 1620 tcgtactgat catgagagac tggctgatactcaaagttgc agttacttag ctgcatgaga 1680 ataatactat tataagttag gtgacaaatgatgttgatta tgtaaggata tacttagcta 1740 cattttcagt cagtatgaac ttcctgatacaaatgtaggg atatatactg tatttttaaa 1800 catttctcac caactttctt atgtgtgttctttttaaaaa ttttttttct tttaaaatat 1860 ttaacagttc aatctcaata agacctcgcattatgtatga atgttattca ctgactagat 1920 ttattcatac catgagacaa cactatttttatttatatat gcatatatat acatacatga 1980 aataaataca tcaatataaa aataaaaaaaaacggaattc 2020 5 445 PRT rat 5 Met Ser Thr Thr Leu Ser Ser Leu Lys GluLys Val Ser Ala Ala Arg 1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys LeuHis Val Lys Pro Asp Val Ser 20 25 30 Glu Leu Met Phe Ala Asp Trp Val GluPhe Ser Pro Tyr Glu Ile Gly 35 40 45 Met Ala Lys Tyr Gly Thr Phe Met ThrPro Asp Leu Phe Gly Ser Lys 50 55 60 Phe Phe Met Gly Thr Val Val Lys LysTyr Glu Glu Asn Pro Leu His 65 70 75 80 Phe Leu Met Gly Val Trp Gly SerAla Phe Ser Ile Leu Phe Asn Arg 85 90 95 Val Leu Gly Val Ser Gly Ser GlnAsn Lys Gly Ser Thr Met Glu Glu 100 105 110 Glu Leu Glu Asn Ile Thr AlaLys His Ile Val Ser Asn Asp Ser Ser 115 120 125 Asp Ser Asp Asp Glu AlaGln Gly Pro Lys Gly Thr Glu Asn Glu Asp 130 135 140 Ala Glu Arg Glu TyrGln Asn Asp Asn Gln Ala Ser Trp Val His Arg 145 150 155 160 Met Leu MetAla Leu Val Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu 165 170 175 Gly ArgAla Gly Lys Glu His Asn Phe Met Leu Gly Leu Asn Leu Asn 180 185 190 ThrSer Tyr Pro Leu Ser Pro Leu Arg Asp Phe Ser Pro Gln Asp Ser 195 200 205Phe Asp Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe 210 215220 Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val 225230 235 240 Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile LeuArg 245 250 255 Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe SerAla Arg 260 265 270 Pro Ser Asp Thr Ser Pro Pro Phe Lys Glu Leu Leu LeuAla Glu Lys 275 280 285 Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys IleAsp Pro Tyr Val 290 295 300 Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr ValPhe Lys Pro Lys Asn 305 310 315 320 Pro Asp Val Glu Lys Asp Cys Pro ThrIle Ile His Phe Val Leu Ala 325 330 335 Asn Ile Asn Phe Arg Lys Tyr LysAla Pro Gly Val Leu Arg Glu Thr 340 345 350 Lys Glu Glu Lys Glu Ile AlaAsp Phe Asp Ile Phe Asp Asp Pro Glu 355 360 365 Ser Pro Phe Ser Thr PheAsn Phe Gln Tyr Pro Asn Gln Ala Phe Lys 370 375 380 Arg Leu His Asp LeuMet Tyr Phe Asn Thr Leu Asn Asn Ile Asp Val 385 390 395 400 Ile Lys AspAla Ile Val Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro 405 410 415 Ser ArgCys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Lys Phe Phe 420 425 430 AsnLys Glu Phe Leu Ser Lys Pro Thr Ala Glu Ser Ile 435 440 445 6 1782 DNArat 6 cgatttggtt agacatatta tttcaaatag cttttatctg tgtccatgtc tatgtattta60 aagccacctt attctttttg tgtgtgtgtg tgaaaagaga atgagtacga ccttgagtag 120cttgaaggaa aaggtcagcg ccgcccggtg tcctctgcct ctcttcacct gtctccatgt 180caaaccggac gtgtcagagc tgatgtttgc cgattgggta gaatttagtc catacgaaat 240tggcatggca aaatatggta cctttatgac tcctgacttg tttggaagca aattttttat 300gggaacagtt gtaaaaaaat atgaagaaaa ccccttgcat ttcttaatgg gtgtctgggg 360cagtgccttt tctatactgt tcaacagagt tttgggagtt tctggctcac agaataaagg 420ttctacaatg gaggaggaat tagaaaatat tacagcaaag cacattgtga gtaacgacag 480ctctgacagc gatgacgagg cccaaggacc caaaggcacc gagaatgaag atgcggaaag 540agagtaccaa aatgacaacc aagcaagttg ggtccatcgg atgctaatgg ccttggtgag 600tgactcagct ttattcaata cccgagaagg acgtgctggg aaggagcata acttcatgtt 660gggcttgaat ctcaacacat cgtatccact gtctcccctg agagacttca gcccccaaga 720ttccttcgat gatgatgaac tcgacgcagc ggtagcagat ccagatgaat ttgaacgaat 780atatgaacca ctggatgtca aaagtaaaaa gattcatgtt gtagacagtg ggctcacgtt 840taacctgccg tatcccttga ttctgcgacc tcagagaggt gtggatctca tcatttcctt 900tgacttttct gcaaggccaa gtgacaccag ccctccattc aaggaacttc tgcttgcaga 960gaagtgggct aaaatgaaca agctcccttt tccaaagatt gatccttacg tgtttgatcg 1020ggaaggattg aaggaatgct atgtgtttaa acctaagaat cctgatgtgg aaaaggattg 1080cccaaccatt atccactttg ttctggccaa catcaacttc agaaagtaca aggccccagg 1140tgttctgagg gaaaccaaag aagagaaaga aatagctgac tttgacattt tcgatgaccc 1200cgaatcgcca ttttcaacct tcaacttcca gtatccaaat caagcattca aaaggctaca 1260tgatctgatg tacttcaaca cactgaacaa cattgatgtg ataaaggatg ccattgttga 1320gagcattgaa tacagaagac agaacccatc tcgttgctct gtttccctca gtaatgttga 1380ggcaagaaaa ttcttcaaca aggagttcct aagtaaaccc acagcggagt ccatttgaat 1440tccatgacta ctggagttca gagccacatg agagactcat cttactatgc acaagagact 1500gactgctact cagagttgct ggggacggag gcgtgtgtta ggtgaaaatg gtgttgatta 1560tgcaatactt ggcaacagtt tctgacagta tgaatttttt gtacataagc atagggctat 1620atactgtatt ttaaacattc ctcacatttt tacctgagca tttttatata tataaaaata 1680tcctttcctt ttataaatat ttaatagtta actcagtaaa aaaaagcttc ccattgtgtg 1740tgaatgttat tctgaactag atttgttcat gccatgttac aa 1782 7 1792 DNA rat 7caattgtttt agaatacaga catctatttc cagggagctt tctttctgtt gtctaatcga 60gaccacagat tgccagaaat aataggactt cgtttcatta taaaaagaga atgagtacga 120ccttgagtag cttgaaggaa aaggtcagcg ccgcccggtg tcctctgcct ctcttcacct 180gtctccatgt caaaccggac gtgtcagagc tgatgtttgc cgattgggta gaatttagtc 240catacgaaat tggcatggca aaatatggta cctttatgac tcctgacttg tttggaagca 300aattttttat gggaacagtt gtaaaaaaat atgaagaaaa ccccttgcat ttcttaatgg 360gtgtctgggg cagtgccttt tctatactgt tcaacagagt tttgggagtt tctggctcac 420agaataaagg ttctacaatg gaggaggaat tagaaaatat tacagcaaag cacattgtga 480gtaacgacag ctctgacagc gatgacgagg cccaaggacc caaaggcacc gagaatgaag 540atgcggaaag agagtaccaa aatgacaacc aagcaagttg ggtccatcgg atgctaatgg 600ccttggtgag tgactcagct ttattcaata cccgagaagg acgtgctggg aaggagcata 660acttcatgtt gggcttgaat ctcaacacat cgtatccact gtctcccctg agagacttca 720gcccccaaga ttccttcgat gatgatgaac tcgacgcagc ggtagcagat ccagatgaat 780ttgaacgaat atatgaacca ctggatgtca aaagtaaaaa gattcatgtt gtagacagtg 840ggctcacgtt taacctgccg tatcccttga ttctgcgacc tcagagaggt gtggatctca 900tcatttcctt tgacttttct gcaaggccaa gtgacaccag ccctccattc aaggaacttc 960tgcttgcaga gaagtgggct aaaatgaaca agctcccttt tccaaagatt gatccttacg 1020tgtttgatcg ggaaggattg aaggaatgct atgtgtttaa acctaagaat cctgatgtgg 1080aaaaggattg cccaaccatt atccactttg ttctggccaa catcaacttc agaaagtaca 1140aggccccagg tgttctgagg gaaaccaaag aagagaaaga aatagctgac tttgacattt 1200tcgatgaccc cgaatcgcca ttttcaacct tcaacttcca gtatccaaat caagcattca 1260aaaggctaca tgatctgatg tacttcaaca cactgaacaa cattgatgtg ataaaggatg 1320ccattgttga gagcattgaa tacagaagac agaacccatc tcgttgctct gtttccctca 1380gtaatgttga ggcaagaaaa ttcttcaaca aggagttcct aagtaaaccc acagcggagt 1440ccatttgaat tccatgacta ctggagttca gagccacatg agagactcat cttactatgc 1500acaagagact gactgctact cagagttgct ggggacggag gcgtgtgtta ggtgaaaatg 1560gtgttgatta tgcaatactt ggcaacagtt tctgacagta tgaatttttt gtacataagc 1620atagggctat atactgtatt ttaaacattc ctcacatttt tacctgagca tttttatata 1680tataaaaata tcctttcctt ttataaatat ttaatagtta actcagtaaa aaaaagcttc 1740ccattgtgtg tgaatgttat tctgaactag atttgttcat gccatgttac aa 1792 8 441 PRTmouse 8 Met Ser Met Thr Leu Ser Ser Leu Lys Glu Lys Val Asn Ala Ala Arg1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro Asp ValSer 20 25 30 Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr Glu IleGly 35 40 45 Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe Gly SerLys 50 55 60 Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn Pro LeuHis 65 70 75 80 Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu PheAsn Arg 85 90 95 Val Leu Gly Val Ser Gly Ser Gln Asn Lys Gly Ser Thr MetGlu Glu 100 105 110 Glu Leu Glu Asn Ile Thr Ala Lys His Ile Val Ser AsnAsp Ser Ser 115 120 125 Asp Ser Asp Asp Glu Ala Gln Gly Pro Lys Gly ThrGlu Asn Glu Glu 130 135 140 Ala Glu Lys Glu Tyr Gln Ser Asp Asn Gln AlaSer Trp Val His Arg 145 150 155 160 Met Leu Met Ala Leu Val Ser Asp SerAla Leu Phe Asn Thr Arg Glu 165 170 175 Gly Arg Ala Gly Lys Val His AsnPhe Met Leu Gly Leu Asn Leu Asn 180 185 190 Thr Ser Tyr Pro Leu Ser ProLeu Arg Asp Phe Ser Ser Gln Asp Ser 195 200 205 Phe Asp Asp Glu Leu AspAla Ala Val Ala Asp Pro Asp Glu Phe Glu 210 215 220 Arg Ile Tyr Glu ProLeu Asp Val Lys Ser Lys Lys Ile His Val Val 225 230 235 240 Asp Ser GlyLeu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg Pro 245 250 255 Gln ArgGly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg Pro 260 265 270 SerAsp Thr Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala Glu Lys Trp 275 280 285Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro Tyr Val Phe 290 295300 Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro Lys Asn Pro 305310 315 320 Asp Val Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val Leu AlaAsn 325 330 335 Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Leu Arg GluThr Lys 340 345 350 Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp AspPro Glu Ser 355 360 365 Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn GlnAla Phe Lys Arg 370 375 380 Leu His Asp Leu Met Tyr Phe Asn Thr Leu AsnAsn Ile Asp Val Ile 385 390 395 400 Lys Asp Ala Ile Val Glu Ser Ile GluTyr Arg Arg Gln Asn Pro Ser 405 410 415 Arg Cys Ser Val Ser Leu Ser AsnVal Glu Ala Arg Lys Phe Phe Asn 420 425 430 Lys Glu Phe Leu Ser Lys ProThr Val 435 440 9 1861 DNA mouse 9 gaaaatgatt tgcttagata tgttattttgaataactttt atctgtgccc catgcctatg 60 tatttaaagc catctcttct tttcttatgtttgtggacag aggatgagca tgaccctgag 120 tagtttgaag gaaaaggtca atgccgcccggtgtcctttg cctctcttca cgtgtctcca 180 cgtcaaacct gatgtgtcag agctgatgtttgccgattgg gtggaattta gtccatatga 240 gattggcatg gcaaaatatg gtacctttatggctcctgac ctatttggaa gcaagttttt 300 tatgggaaca gttgtaaaaa aatatgaagaaaaccccttg catttcttga tgggtgtctg 360 gggcagtgcc ttttctatac tgttcaacagagttttggga gtttctggct cacagaataa 420 aggctctaca atggaagagg aattagaaaatattacagca aagcacatcg tgagtaatga 480 cagctccgac agtgatgatg aggctcaaggacccaaaggc accgagaatg aagaagctga 540 aaaagagtac caaagcgaca accaagcaagttgggtccat cggatgctaa tggccttggt 600 gagcgactcg gctttattca atacccgagaaggacgtgcc ggaaaggtgc ataacttcat 660 gctgggcttg aatctcaaca catcatatccactgtctccc ctgagagact tcagctctca 720 ggattccttc gatgacgagc tcgacgcagcggtagcagat ccagatgaat ttgaacgaat 780 atatgaacca ctggatgtca aaagtaagaagattcatgtg gtagatagtg ggctcacatt 840 taacctgcca tatcccttga ttcttcgacctcagagaggt gtggatctta tcatctcctt 900 tgacttttct gcaaggccga gtgacaccagtccccctttc aaggaacttc tgcttgcaga 960 gaagtgggcg aaaatgaaca agcttccctttccaaagatc gatccttatg tgtttgatcg 1020 ggaaggatta aaggaatgct atgtttttaaacctaagaat cctgatgtgg agaaggattg 1080 cccaaccatt atccactttg ttctggccaacatcaacttc agaaagtaca aggccccagg 1140 tgttctaagg gaaaccaaag aagagaaagaaattgctgac tttgacattt ttgatgaccc 1200 cgaatcgcca ttttcaacct tcaactttcagtatcccaat caagcattca aaaggcttca 1260 cgatttgatg tacttcaaca cactgaacaacattgatgtg ataaaggatg ccattgttga 1320 gagcattgaa tacagaagac agaacccatctcgttgctct gtttccctca gtaatgttga 1380 agcaagaaaa ttcttcaata aggagtttctaagtaaaccc actgtgtaat ttctgtgctg 1440 ggatgatcaa gccatttgaa ttccatgacaatttgagttc agaagacatt agaggtcatc 1500 ttactatgca gaagagactg gctgctactcaaagttgtgg agatttagcc atgtgttagg 1560 tgaaaatgat gttgattatg taatacttagcaacagtttc tgacagtatg aattttttga 1620 cattagcata gagctatata ctgtattttaaacattcctc acatttttta cctgtacttt 1680 ttatataaat atgacatgtc ttttcttttgaaaatattta atagtttaac tcagtaaagg 1740 agacttccca ttgtgtgtga atgttattctgaactagatt tgttcatgcc atgttacaac 1800 actattttta tttaaatgtt tatatttacacatacgaaat aaatactttg ctgtacaaat 1860 t 1861 10 1860 DNA mouse 10agttgtttta aaatacacac atctttttcc ctggaacttt atttctgttg tctacttgag 60accacagatt tccaggaata ataggacttc atttcattaa ggatgagcat gaccctgagt 120agtttgaagg aaaaggtcaa tgccgcccgg tgtcctttgc ctctcttcac gtgtctccac 180gtcaaacctg atgtgtcaga gctgatgttt gccgattggg tggaatttag tccatatgag 240attggcatgg caaaatatgg tacctttatg gctcctgacc tatttggaag caagtttttt 300atgggaacag ttgtaaaaaa atatgaagaa aaccccttgc atttcttgat gggtgtctgg 360ggcagtgcct tttctatact gttcaacaga gttttgggag tttctggctc acagaataaa 420ggctctacaa tggaagagga attagaaaat attacagcaa agcacatcgt gagtaatgac 480agctccgaca gtgatgatga ggctcaagga cccaaaggca ccgagaatga agaagctgaa 540aaagagtacc aaagcgacaa ccaagcaagt tgggtccatc ggatgctaat ggccttggtg 600agcgactcgg ctttattcaa tacccgagaa ggacgtgccg gaaaggtgca taacttcatg 660ctgggcttga atctcaacac atcatatcca ctgtctcccc tgagagactt cagctctcag 720gattccttcg atgacgagct cgacgcagcg gtagcagatc cagatgaatt tgaacgaata 780tatgaaccac tggatgtcaa aagtaagaag attcatgtgg tagatagtgg gctcacattt 840aacctgccat atcccttgat tcttcgacct cagagaggtg tggatcttat catctccttt 900gacttttctg caaggccgag tgacaccagt ccccctttca aggaacttct gcttgcagag 960aagtgggcga aaatgaacaa gcttcccttt ccaaagatcg atccttatgt gtttgatcgg 1020gaaggattaa aggaatgcta tgtttttaaa cctaagaatc ctgatgtgga gaaggattgc 1080ccaaccatta tccactttgt tctggccaac atcaacttca gaaagtacaa ggccccaggt 1140gttctaaggg aaaccaaaga agagaaagaa attgctgact ttgacatttt tgatgacccc 1200gaatcgccat tttcaacctt caactttcag tatcccaatc aagcattcaa aaggcttcac 1260gatttgatgt acttcaacac actgaacaac attgatgtga taaaggatgc cattgttgag 1320agcattgaat acagaagaca gaacccatct cgttgctctg tttccctcag taatgttgaa 1380gcaagaaaat tcttcaataa ggagtttcta agtaaaccca ctgtgtaatt tctgtgctgg 1440gatgatcaag ccatttgaat tccatgacaa tttgagttca gaagacatta gaggtcatct 1500tactatgcag aagagactgg ctgctactca aagttgtgga gatttagcca tgtgttaggt 1560gaaaatgatg ttgattatgt aatacttagc aacagtttct gacagtatga attttttgac 1620attagcatag agctatatac tgtattttaa acattcctca cattttttac ctgtactttt 1680tatataaata tgacatgtct tttcttttga aaatatttaa tagtttaact cagtaaagga 1740gacttcccat tgtgtgtgaa tgttattctg aactagattt gttcatgcca tgttacaaca 1800ctatttttat ttaaatgttt atatttacac atacgaaata aatactttgc tgtacaaatt 186011 1966 DNA mouse 11 agttgtttta aaatacacac atctttttcc ctggaactttatttctgttg tctacttgag 60 accacagatt tccaggaata ataggacttc atttcattataaaatgaaaa tgatttgctt 120 agatatgtta ttttgaataa cttttatctg tgccccatgcctatgtattt aaagccatct 180 cttcttttct tatgtttgtg gacagaggat gagcatgaccctgagtagtt tgaaggaaaa 240 ggtcaatgcc gcccggtgtc ctttgcctct cttcacgtgtctccacgtca aacctgatgt 300 gtcagagctg atgtttgccg attgggtgga atttagtccatatgagattg gcatggcaaa 360 atatggtacc tttatggctc ctgacctatt tggaagcaagttttttatgg gaacagttgt 420 aaaaaaatat gaagaaaacc ccttgcattt cttgatgggtgtctggggca gtgccttttc 480 tatactgttc aacagagttt tgggagtttc tggctcacagaataaaggct ctacaatgga 540 agaggaatta gaaaatatta cagcaaagca catcgtgagtaatgacagct ccgacagtga 600 tgatgaggct caaggaccca aaggcaccga gaatgaagaagctgaaaaag agtaccaaag 660 cgacaaccaa gcaagttggg tccatcggat gctaatggccttggtgagcg actcggcttt 720 attcaatacc cgagaaggac gtgccggaaa ggtgcataacttcatgctgg gcttgaatct 780 caacacatca tatccactgt ctcccctgag agacttcagctctcaggatt ccttcgatga 840 cgagctcgac gcagcggtag cagatccaga tgaatttgaacgaatatatg aaccactgga 900 tgtcaaaagt aagaagattc atgtggtaga tagtgggctcacatttaacc tgccatatcc 960 cttgattctt cgacctcaga gaggtgtgga tcttatcatctcctttgact tttctgcaag 1020 gccgagtgac accagtcccc ctttcaagga acttctgcttgcagagaagt gggcgaaaat 1080 gaacaagctt ccctttccaa agatcgatcc ttatgtgtttgatcgggaag gattaaagga 1140 atgctatgtt tttaaaccta agaatcctga tgtggagaaggattgcccaa ccattatcca 1200 ctttgttctg gccaacatca acttcagaaa gtacaaggccccaggtgttc taagggaaac 1260 caaagaagag aaagaaattg ctgactttga catttttgatgaccccgaat cgccattttc 1320 aaccttcaac tttcagtatc ccaatcaagc attcaaaaggcttcacgatt tgatgtactt 1380 caacacactg aacaacattg atgtgataaa ggatgccattgttgagagca ttgaatacag 1440 aagacagaac ccatctcgtt gctctgtttc cctcagtaatgttgaagcaa gaaaattctt 1500 caataaggag tttctaagta aacccactgt gtaatttctgtgctgggatg atcaagccat 1560 ttgaattcca tgacaatttg agttcagaag acattagaggtcatcttact atgcagaaga 1620 gactggctgc tactcaaagt tgtggagatt tagccatgtgttaggtgaaa atgatgttga 1680 ttatgtaata cttagcaaca gtttctgaca gtatgaattttttgacatta gcatagagct 1740 atatactgta ttttaaacat tcctcacatt ttttacctgtactttttata taaatatgac 1800 atgtcttttc ttttgaaaat atttaatagt ttaactcagtaaaggagact tcccattgtg 1860 tgtgaatgtt attctgaact agatttgttc atgccatgttacaacactat ttttatttaa 1920 atgtttatat ttacacatac gaaataaata ctttgctgtacaaatt 1966 12 560 DNA Human 12 taattcattt caatgatgta aagattttgaatgtgtgagg aagtgctttt gtattccttt 60 tctctggaaa aaaaaaaaaa aaaaaaaattcacattttaa cccttaactg cccattccct 120 ccaagaatgg taacattttt agatgaggaagaatgaagtt tgcctgaata gagtcaagaa 180 aggaagggga tcgcatagaa cagactcgcttgatgcatga ttgcattgat gtttcgttga 240 agataaagca gaggagcgcc tgtgacagggagtccagggg ctaagtttct tccaggctcc 300 acagttgcta attcattctc cagttcagatgtagacatat aatctagagt tatgattatt 360 ttttaaatga agatagttac ttccatagagcttatttttt gttgttcatt caggacctag 420 taatttctag aagtaataag acttatttttattataaagt tataagattt tgattggaag 480 tactattttg aatagcattc tttctgtgtctgtttataaa tttaaagtca tctttttctt 540 tcttctgtgg acagagaatg 560 13 667DNA rat 13 taaaaaaagt aatatttagg tgagatggta agactatgca ttgcttttgagggggatgtg 60 agttcagtag tcagcaccta catctggcag cgcacaactg cctgtaactccagctttagg 120 aggagccgat acctctggcc tttatgaaca cctacactca catgcatatatccacacaca 180 gataatatac atatgcatat tttacttttt atattcatat tttaaaataatagaagtggt 240 agaaaaaata ctctttgcct agtaagagtc aaataaggaa atggatcataggaaacaaat 300 gtacttgatg tgtcaccaga gggtgacatt tcatctgaag ataaagcaggagagacggga 360 caacctgtgc cagggacacc agctgagaga attagttccc agaactatagctgccaaatc 420 ttcccccact tcaaatgttg acacagtccc cagagattca attgttttagaatacagaca 480 tctatttcca gggagctttc tttctgttgt ctaatcgaga ccacagattgccagaaataa 540 taggacttcg tttcattata aaaaggcaag cgatttggtt agacatattatttcaaatag 600 cttttatctg tgtccatgtc tatgtattta aagccacctt attctttttgtgtgtgtgtg 660 tgaaaag 667 14 739 DNA mouse base 496 n = a, g, c, t,unknown, or other 14 tggtaagatg gtgaatagaa gtgctattta ggtgagatcgtgaatacaag tgatattaaa 60 agcaggaagg aggaggtttt cgctcctcag cagataagcccatgcactgc tcttgtgggg 120 acatgagttc aacaaccagc acctatgtct gacagctcataactacctgt aattccagat 180 tcaggaggtg ccaatacctc tggcctctgt gaatatctgcactcacaatc atatatccac 240 acacagatac atattcatat acatatttta ttttttatattcacatttta aaataataaa 300 ttgaaatggt agaagaacac tctttgcctc ataagagacaaataaggaaa tggaccatgg 360 gaaacaaatg gacttgatgt gtcaccagag ggtgacatttcatctgaaga taaagcaggg 420 gagaaaggac agctgtgcca gggaacgcca gctgagagaactagttctca acactctagt 480 tgccaaatct tcctcnnctt caaatgttga cacagtcttcagagattcag ttgttttaaa 540 atacacacat ctttttccct ggaactttat ttctgttgtctacttgagac cacagatttc 600 caggaataat aggacttcat ttcattataa aatgaaaatgatttgcttag atatgttatt 660 ttgaataact tttatctgtg ccccatgcct atgtatttaaagccatctct tcttttctta 720 tgtttgtgga cagaggatg 739

1. A phospholipase A2 which is calcium-independent.
 2. The phospholipaseA2 according to claim 1, wherein it is specific to hippocampus.
 3. Thephospholipase A2 according to claim 1 or 2, wherein it is acalcium-independent phospholipase A2 and has an amino acid sequencedescribed in SEQ ID NO: 1, NO: 5 or NO: 8 of the Sequence Listing or anamino acid sequence where one or more amino acid(s) therein is/aresubstituted with other amino acid(s) or deleted therefrom or one or moreamino acid(s) is/are added thereto.
 4. A gene having a base sequencewhich codes for the phospholipase A2 described in any of claims 1 to 3.5. The gene according to claim 4, wherein the gene is DNA.
 6. Anantibody where the full length or the fragment of the phospholipase A2described in any of claims 1 to 3 is an antigen.
 7. The antibodyaccording to claim 6, wherein the antibody is a monoclonal antibody. 8.A gene which has a base sequence existing in intron where the basesequence is able to make the initiation of transcription of RNA by anexternal stimulation possible.
 9. The gene according to claim 8, whereinthe initiation of transcription of RNA is site-specific.
 10. The geneaccording to claim 8 or 9, wherein the external stimulation is a kainicacid stimulation or an electric stimulation.
 11. The gene according toany of claims 8 to 10, wherein the gene has a base sequence described inSEQ ID NO: 12, NO: 13 or NO: 14 of the Sequence Listing or has a basesequence comprising a partial sequence where a part thereof is deleted,added or substituted.
 12. A promoter which is a base sequence existingin intron and able to make the initiation of transcription of RNA by anexternal stimulation possible.
 13. The promoter according to claim 12,wherein the initiation of transcription of RNA is site-specific.
 14. Thepromoter according to claim 12 or 13, wherein the external stimulationis a kainic acid stimulation or an electric stimulation.
 15. Aregulatory gene having a regulatory element on the upper stream of thepromoter described in claims 12-14.
 16. A process where the genedescribed in any of claims 8-15 is introduced into the upper stream ofgene coding for protein to initiate the transcription of RNA by anexternal stimulation whereby the said protein is expressed in responseto the external stimulation.
 17. The process according to claim 16,wherein the gene coding for the protein is CRE.
 18. The processaccording to claim 16, wherein the gene coding for the protein is a genewhich codes for a toxin protein.
 19. The process according to claim 18,wherein the toxin protein is diphtheria toxin.
 20. A living thing wherethe gene described in any of claims 8-15 is introduced into the upperstream of a gene coding for protein.
 21. The living thing according toclaim 20, wherein the living thing is an experimental animal.
 22. Theliving thing according to claim 20 or 21, wherein the gene coding forprotein is CRE.
 23. A method for detection or identification of nervestem cells where nerve cells are stimulated by an external stimulationand expression of mRNA coding for the phospholipase A2 described in anyof claims 1 to 3 is detected or identified.
 24. The method according toclaim 23, wherein the external stimulation is a kainic acid stimulationor an electric stimulation.